Kitto G B, Smith G, Thiet T Q, Mason M, Davidson L
J Bacteriol. 1979 Jan;137(1):204-12. doi: 10.1128/jb.137.1.204-212.1979.
Two enzymes that catalyze the hydrolysis of l-asparagine have been isolated from extracts of Pseudomonas geniculata. After initial salt fractionation, the enzymes were separated by chromatography on diethylaminoethyl-Sephadex and purified to homogeneity by gel filtration, ion-exchange chromatography, and preparative polyacrylamide electrophoresis. The enzymes differ markedly in physicochemical properties. One enzyme, termed asparaginase A, has a molecular weight of approximately 96,000 whereas the other, termed asparaginase AG, has a molecular weight of approximately 135,000. Both enzymes are tetrameric. The asparaginase A shows activity only with l-asparagine as substrate, whereas the asparaginase AG hydrolyzes l-asparagine and l-glutamine at approximately equal rates and it is also active with d-asparagine and d-glutamine as substrates. The asparaginase A was found to be devoid of antitumor activity in mice, whereas the asparaginase AG was effective in increasing the mean survival times of both C3H mice carrying the asparagine-requiring Gardner 6C3HED tumor line and Swiss mice bearing the glutamine-requiring Ehrlich ascites tumor line. These differences in antitumor activity were related to differences in the K(m) values for l-asparagine for the two enzymes. The asparaginase A has a K(m) value of 1 x 10(-3) M for this substrate whereas the corresponding value for the AG enzyme is 1.5 x 10(-5) M. Thus the concentration of asparagine necessary for maximal activity of the asparaginase A is very high compared with that of the normal plasma level of asparagine, which is approximately 50 muM.
从膝状假单胞菌提取物中分离出了两种催化L-天冬酰胺水解的酶。经过初步盐分级分离后，通过二乙氨基乙基-葡聚糖凝胶柱色谱法分离这些酶，并通过凝胶过滤、离子交换色谱法和制备性聚丙烯酰胺电泳将其纯化至均一。这两种酶在物理化学性质上有显著差异。一种酶称为天冬酰胺酶A，分子量约为96,000，而另一种称为天冬酰胺酶AG，分子量约为135,000。两种酶均为四聚体。天冬酰胺酶A仅以L-天冬酰胺作为底物表现出活性，而天冬酰胺酶AG以大致相同的速率水解L-天冬酰胺和L-谷氨酰胺，并且它对D-天冬酰胺和D-谷氨酰胺作为底物也有活性。发现天冬酰胺酶A在小鼠中没有抗肿瘤活性，而天冬酰胺酶AG有效地延长了携带需要天冬酰胺的Gardner 6C3HED肿瘤系的C3H小鼠和携带需要谷氨酰胺的艾氏腹水肿瘤系的瑞士小鼠的平均存活时间。这些抗肿瘤活性的差异与两种酶对L-天冬酰胺的米氏常数（K(m)）值的差异有关。天冬酰胺酶A对该底物的K(m)值为1×10⁻³M，而AG酶的相应值为1.5×10⁻⁵M。因此，与正常血浆中天冬酰胺水平（约50μM）相比，天冬酰胺酶A达到最大活性所需的天冬酰胺浓度非常高。
